Background: Pulmonary arterial hypertension (PAH) is a deadly vascular disease with increased prevalence with human immunodeficiency virus (HIV) infection. HIV-PAH may be even more prevalent than originally anticipated, yet diagnostics and treatments are limited for this enigmatic form of PAH. Recently, we have found that vascular stiffening and glutamine metabolism are linked processes in PAH, including primate and human examples of HIV-PAH. The transcriptional co-activators YAP/TAZ induce the microRNA (miR)-130/301 family and glutaminase (GLS1) to control these phenotypes. In PAH rodent models, inhibition of miR-130/301, YAP, or GLS1 improve PAH. We also found that HIV-infected T cells release cell-free miR-21 molecules and up-regulate glutaminolytic and matrix remodeling pathways in co-cultured pulmonary vascular cells. We hypothesize that a YAP/TAZ-miR-130/301-GLS1 axis is induced by a miR-21-mediated process, linking HIV-infected T cells and pulmonary vascular cells and thus activating vascular stiffening, glutaminolysis, and HIV-PAH. Specific Aims: Aim 1) Determine if miR-21 released from HIV-infected T cells induces pulmonary vascular metabolic dysfunction and matrix remodeling. In vitro, we will investigate the direct delivery and actions of miR-21 to pulmonary vascular cells from HIV-infected T cells. In vivo, employing transfusions of miR-21-replete vs. miR-21-depleted plasma into miR-21-/- mice vs. wildtype mice, we will determine if circulating miR-21 is delivered to pulmonary endothelium and induces vascular stiffening, glutaminolysis, and PAH. Results could establish an entirely novel miR link between HIV infection and pulmonary vascular glutaminolysis and stiffness. Aim 2) Determine if pulmonary arterial stiffness and glutaminolysis are evident in humans with HIV-PAH. From prior collected HIV-PAH samples, we will correlate plasma miR-21 levels with pulmonary artery (PA) compliance as calculated from hemodynamic data and plasma metabolites reflective of PA glutaminolysis. Using optimized techniques based on our published protocols, we will also assess for activation of the YAP/TAZ-miR- 130/301-GLS1 axis in PA endothelial cells collected via catheterization of HIV-PAH patients. These results could establish this mechanism in human HIV-PAH and suggest needed molecular diagnostics for HIV-PAH detection. Aim 3) Determine if up-regulation of GLS1 is necessary for promoting SIV-PAH. In SIV-PAH macaques, we will administer CB-839, an oral GLS1 inhibitor being tested in human cancer trials, to determine its effects on stiffness, glutaminolysis, and PAH. Results could demonstrate the direct pathogenic actions of GLS1 in HIV- relevant PAH and thus could re-purpose this drug for rapid, expedited trials in human HIV and PAH patients. Significance: Our team is uniquely positioned for making major molecular discoveries of HIV-PAH. We will leverage the only known reliable animal model of HIV-PAH with human studies, ensuring mechanistic insight and applicability to human disease to an extent never possible before. Perhaps most importantly, it offers a rare opportunity to establish a much needed targeted therapeutic for this historically neglected vascular disease.